FAK loss reduces BRAFV600E-induced ERK phosphorylation to promote intestinal stemness and cecal tumor formation

BRAFV600E mutation is a driver mutation in the serrated pathway to colorectal cancers. BRAFV600E drives tumorigenesis through constitutive downstream extracellular signal-regulated kinase (ERK) activation, but high-intensity ERK activation can also trigger tumor suppression. Whether and how oncogenic ERK signaling can be intrinsically adjusted to a ‘just-right’ level optimal for tumorigenesis remains undetermined. In this study, we found that FAK (Focal adhesion kinase) expression was reduced in BRAFV600E-mutant adenomas/polyps in mice and patients. In Vil1-Cre;BRAFLSL-V600E/+;Ptk2fl/fl mice, Fak deletion maximized BRAFV600E’s oncogenic activity and increased cecal tumor incidence to 100%. Mechanistically, our results showed that Fak loss, without jeopardizing BRAFV600E-induced ERK pathway transcriptional output, reduced EGFR (epidermal growth factor receptor)-dependent ERK phosphorylation. Reduction in ERK phosphorylation increased the level of Lgr4, promoting intestinal stemness and cecal tumor formation. Our findings show that a ‘just-right’ ERK signaling optimal for BRAFV600E-induced cecal tumor formation can be achieved via Fak loss-mediated downregulation of ERK phosphorylation.


Introduction
Colorectal cancer (CRC) is a heterogeneous disease arising through several discrete evolutionary pathways.The best-known and most-studied pathway to CRC is the canonical pathway, in which cancer originates from conventional adenomatous polyps bearing APC (adenomatous polyposis coli) mutation 1,2 .Recently a new "alternative" pathway through serrated adenoma-the serrated pathway -has been uncovered.Mice studies have established that the BRAF V600E mutation is a driver mutation in the serrated pathway [3][4][5] .In patients, BRAF V600E mutation is found in 50-67% of serrated CRC 6 and 10-15% of all CRCs 7 .
The "Goldilocks principle" applies to mutant APC-driven and mutant BRAF-driven intestinal tumorigenesis: a threshold of oncogenic signaling needs to be reached for dysplastic lesions to form, but optimum tumor development requires "just-right" levels of oncogenic signaling, with too much being as detrimental as too little.In the canonical pathway to CRC, the primary driving force is mutant APCmediated activation of Wnt/b-catenin signaling 8 , and the "just-right" level of Wnt/b-catenin signaling optimal for tumor formation is achieved mainly by the selection for speci c APC mutant proteins based on their residual b-catenin-downregulating activity [9][10][11][12] .The selection for APC mutations in the intestine is in uenced by the underlying basal/physiological level of Wnt activity and stemcell number, and APC mutation spectra vary throughout the intestinal tract resulting in different APC mutation spectra in the proximal and distal CRCs 10,11 .In addition to the different mutation spectra, the 'optimal' thresholds for proximal and distal cancers are also variable 11 .
BRAF V600E drives tumorigenesis through constitutive downstream ERK1/2 activation 13 , but hyperactivation of ERK induced by oncogenic BRAF V600E is not tolerated in the intestine: high ERK activation, induced by transgenic expression of oncogenic BRAF (BRAF V600K ) or by activation of two BRAF alleles in BRAF V600E/V600E mutant mice, engages tumor suppressive mechanisms, causing loss of stem cells and induction of differentiation and senescence 14,15 .Lowering ERK activation by treatment with ERK or MEK (mitogen-activated protein kinase kinase) inhibitor counteracted BRAF V600E -induced organoid disintegration 14,16 .It is therefore presumed that maintaining ERK activation within a narrow threshold range to avoid engaging tumor suppression is pivotal for mutant BRAF to exhibit the strongest transforming activity.However, despite being highly anticipated 16 , the existence of in vivo intrinsic netuning of mutant BRAF-induced ERK activation has never been experimentally examined.Given that over 60 mutations have now been identi ed in BRAF 13,17 , theoretically, mutation selection could be a way to achieve optimal ERK activation.However, because the V600E mutation accounts for about 90% of BRAF mutation seen in human cancer 18 , mutation selection is not the primary means to achieve the "just-right" levels of oncogenic ERK signaling.Normally, ERK activation is self-limiting by the rapid inactivation of upstream kinases and delayed induction of dual-speci c MAKP phosphatases (MKPs/DUSPs) 19 .
Although feedback inhibitors of ERK signaling, including DUSPs are overexpressed in BRAF V600E expressing cells, the ERK signaling pathway is refractory to upstream feedback inhibition 20 .EGFR is a core receptor upstream of the MAPK kinase axis.In vitro cell culture studies show that all activating BRAF mutants are RAS-independent 21 : neither RAS inhibition 21 nor EGFR inhibition 22,23 was able to inhibit mutant-BRAF-induced ERK phosphorylation in BRAF-mutant human CRC cell lines.
In this study, we addressed whether BRAF V600E -induced ERK activation is still tuneable during tumorigenesis in vivo.If yes, what are the factors involved in the regulation?Can BRAF V600E -induced ERK activation be ne-tuned to a "just-right" level optimal for tumor initiation?Our study identi ed FAK as a key regulator of BRAF V600E -induced ERK activation in mutant BRAF-induced serrated tumor formation/initiation and revealed that FAK loss allows BRAF V600E -induced ERK signaling to reach the permissive threshold "just-right" for cecal tumors to form.

Results
FAK expression is reduced in BRAF V600E -mutant serrated lesions in humans and mice.FAK is a cytoplasmic non-receptor tyrosine kinase involved in many aspects and types of cancer 24 .To determine the role of FAK in mutant BRAF-induced serrated CRC, we rst evaluated FAK protein expressions in human BRAF V600E -mutated serrated tumors (11 cases).We examined tissue sections containing BRAF V600E -mutant CRCs, sessile serrated adenoma/polyps (SSA/P)s, and adjacent histologically normal colon from the same tissue block.Results of immunohistochemistry (IHC) staining showed that FAK protein levels were lower in SSA/Ps (5/5) than in normal intestines and CRCs (5/5) (Fig. 1a).FAK expression was more complex in CRCs.FAK levels in CRCs were either similar to (6/11) or lower (4/11) or higher (1/11) than that of the normal intestines (Fig. 1a, b).FAK was mainly localized in the cytoplasm (Fig. 1b).In mice, compared to the neighboring normal mucosa or stroma in the tumor, Fak protein levels were substantially decreased in carcinomas in the colon (Fig. 1c) and adenomas/polyps in the small intestine (SI) (Fig. 1d) in Vill-Cre;BRAF V600E/+ (BC) mice.The downregulation of FAK in human and mouse polyps suggests that FAK loss may play a role in BRAF V600E -induced tumor formation/initiation. Fak deletion promotes BRAF V600E -induced cecal tumor formation.Previous mice studies show that Fak deletion suppresses mammary tumorigenesis 25,26 , mutant Apc-induced intestinal tumorigenesis 27 , skin tumor formation 28 , and hepatocarcinogenesis 29 .To address the functional signi cance of FAK downregulation in BRAF V600E -induced serrated tumor formation/initiation, we generated the Vill-Cre;BRAF V600E/+ ;Fak / (FBC) mice.The Cre-mediated recombination e ciency was con rmed by tdTomato-reporter expression in intestinal crypts in Vill-Cre;Rosa26 LSL-tdTomato/+ mice (Supplementary Fig. 1a).Deletion of Fak in the intestinal epithelium was further con rmed by IHC staining of the intestine in FBC mice (Supplementary Fig. 1b).
Similar to that seen in BC mice, compared to the BRAF V600E/+ (B) mice, the FBC mice exhibited hyperplasia throughout the intestine (Fig. 2a) and thickened small and large intestines (Supplementary Fig. 1c).In BC mice, intestinal tumors were primarily developed in the small intestine at nine months or older (Fig. 2b).Fak loss had minimal impact on tumor incidence in the small intestine and the colon; however, it greatly enhanced BRAF V600E -induced cecal tumor formation: cecal tumor incidence increased from 0% (0/15) in 9-month or older BC mice to 100% (16/16) in FBC mice (Fig. 2c).Cecal adenoma/polyp started to develop in 3-month FBC mice and after six months, all mice (4/4) developed cecal tumors and 25% of the tumors (1/4) were carcinomas (Fig. 2c, d).At nine months or older, 100% of the mice developed cecal tumors with a high incidence (13/16) of carcinoma (Fig. 2c, d, and Supplementary Fig. 1d).IHC staining con rmed that while the stroma showed strong Fak staining, tumor cells were Fak negative (Fig. 2e), hence validating that tumors were originated from Fak-deleted epithelial cells.Of note, no tumor metastasis was found in FBC mice.FBC mice were aged up to 434 days, and the life span of FBC mice was similar to that of BC mice.
Together, these results revealed that Fak deletion promotes, rather than inhibits, BRAF V600E -induced cecal tumor formation.BRAF-mutant CRCs are primarily located in the right colon, including the cecum 30 .The same primary tumor location suggests that the FBC model truthfully recapitulates human BRAF-mutant serrated CRCs, at least by location.
The molecular feature of the cecal tumors in FBC mice closely resembles human SSA/Ps.To characterize the molecular signatures of the cecal tumor in FBC mice, we performed whole-exome sequencing on paired tumors (n=2) and neighboring mucosa.No additional driver mutations were detected in the cecal tumors (Supplementary Table 1), implying that cecal tumor formation in FBC mice does not require additional driver mutations.To evaluate the relevance of FBC cecal tumors to humans, we performed RNA-sequencing (RNA-seq) and Gene Set Enrichment Analysis (GSEA) to determine whether FBC cecal tumors exhibited similar gene expression signatures as human SSA/Ps 31 .The results showed that upregulated genes in human SSA/Ps were signi cantly enriched in cecal tumors in FBC mice (Fig. 3a).Downregulated genes in human SSA/P were also reduced in FBC tumors (Fig. 3b).Together, these results suggest that the FBC cecal tumors greatly resemble human serrated lesions at the molecular level.About 50% of BRAF-mutated CRCs exhibit defective DNA mismatch repair 18 .The results of microsatellite instability (MSI) analysis indicated that most FBC cecal tumors were microsatellite stable (MSS) (Fig. 3c).
It has been shown that mismatch repair de ciency accelerates BRAF-driven serrated tumorigenesis 32 .
Maximizing the oncogenic activity of BRAF V600E without mismatch repair gene mutation and additional driver mutations suggests that in FBC mice, Fak loss created a "just-right" environment optimal for MSS serrated cecal tumor to form.
Fak loss increases intestinal stemness by upregulating Lgr4 levels in FBC mice.We explored the molecular mechanism underlying Fak loss-enhanced cecal tumor formation.Consistent with a prior report 27 , we did not detect any abnormalities in the intestine in Vill-Cre; Fak / mice, implying that FAK loss by itself is not a driving force for intestinal tumorigenesis.A prior study showed that upon TGFβ (transforming growth factor β) receptor inactivation, BRAF V600E -induced right-sided tumorigenesis is supported by microbial-driven in ammation 33 .To test the role of in ammation in FBC tumor formation, we compared sub-cryptal proprial neutrophil in ltration using myeloperoxidase (MPO) as a neutrophil marker for IHC staining.The results showed that, consistent with prior ndings 33 , the number of MPOpositive cells was signi cantly higher in BC mice than in B mice; however, Fak loss did not further increase neutrophil in ltration in FBC mice (Supplementary Fig. 2a).Consistent with this, GSEA results showed that there was no difference in the expression of in ammatory response genes 34 in FBC mice and BC mice (Supplementary Fig. 2b).Together, these ndings imply that Fak loss promotes tumor formation not by enhancing intestinal in ammation.
We next evaluated the roles of cellular senescence, apoptosis, cell proliferation, and Lgr5 expression in cecal tumorigenesis in FBC mice.The results indicated that BRAF V600E was insu cient to trigger senescence evaluated by SA-β-galactosidase staining or apoptosis evaluated by the TUNEL staining in BC mice (Supplementary Fig. 2c, d).Bromodeoxyuridine (BrdU) incorporation assays con rmed mutant BRAF-induced hyperproliferation.However, Fak loss did not further enhance the BrdU incorporation rate (Supplementary Fig. 2e).These results indicated that Fak deletion promotes tumor formation not through modulating cellular senescence, apoptosis, and cell proliferation.
Given that BRAF V600E drives tumorigenesis through constitutive downstream ERK1/2 activation 13 , we examined the impact of Fak loss on ERK pathway transcriptional output.GSEA analysis showed that ERK pathway output was signi cantly increased in BC mice (Fig. 4a), which was consistent with the earlier report 20 , but Fak loss did not further enhance it (Fig. 4f).Wnt pathway activation 32 and activation of transcription co-factor YAP have been implied in BRAF V600E -induced serrated tumorigenesis 33 .In this study, our GSEA results also showed that the expression of intestinal Wnt signature genes 35 and YAP target genes 36 were signi cantly higher in BC mice than in B mice (Fig. 2b, c).Again, Fak loss did not further enhance the activations (Fig. 2g, h).Together, these ndings excluded the possibility that Fak loss promotes cecal tumor formation by enhancing ERK pathway output and activation of the Wnt and YAP pathways.
BRAF V600E poorly initiates colon cancer in mice due to oncogenic BRAF-induced tissue differentiation and loss of intestinal stem cells 15 .With this, GSEA results showed increased expressions of intestinal differentiation signature genes 37 (Fig. 4d) and decreased expressions of intestinal stem cell signature genes 38 (Fig. 4e) in BC mice.Fak deletion did not reverse BRAF V600E -induced tissue differentiation (Fig. 4i) but signi cantly enhanced intestinal stemness (Fig. 4j).These results revealed that Fak deletion promotes BRAF V600E -induced cecal tumor formation through increasing intestinal stemness.
The adult stem cell marker Lgr5 and its relative Lgr4 are R-spondin receptors mediating R-spondin signaling and are critical for intestinal stemness 39,40 .Mutant BRAF reduces Lgr5 expression in the intestinal crypt 15,33 .Our results con rmed the downregulation of Lgr5 in the cecum crypt in BC mice, and we found that Fak loss did not restore Lgr5 expression in FBC mice (Supplementary Fig. 2f).These results thus excluded the possibility that Lgr5 mediates Fak loss-induced intestinal stemness.
Prior studies show that the fetal type of intestinal stem cells has a strikingly different transcriptome than that of adult intestinal stem cells, and the receptor LGR4, but not LGR5, is essential for the cells 41 .In VillinCre ER ;Braf LSL-V600E/+ ;Alk5 / mice, the proximal colonic tumors exhibit fetal intestinal signature 33 .
Consistent with the notion that mutant BRAF-driven right-sided colonic tumors are fetal progenitor phenotypes, GSEA results con rmed enrichment of the fetal-type transcriptomic signatures 41 in cecal mucosa in BC mice.The fetal signature was further enriched in FBC mice (Fig. 4k).Accordingly, the immunoblotting analysis showed that the protein level of Lgr4 was increased in the intestine epithelium in FBC mice (Fig. 4i).Consistent with the fact that intestinal Lgr5 expression was low in FBC mice (Supplementary Fig. 2f), FBC tumors mainly expressed Lgr4 but not Lgr5.In contrast, BC and Apc min/+ tumors expressed both Lgr5 and Lgr4 (Fig. 4m).These results suggest that upregulated Lgr4 mediated the intestinal stemness increase in FBC mice.
FAK loss downregulates EGFR-dependent ERK phosphorylation to increase Lgr4 mRNA expression and protein stability.We addressed how Fak loss mediates Lgr4 increase.A prior study suggested that Wnt signaling maintains quiescent intestinal stem cell pools through suppression of the MAPK pathway in the intestine 42 .Given the fact that Fak loss did not jeopardize ERK pathway transcriptional output (Fig. 4f), Fak loss may increase intestinal stemness by inhibiting ERK phosphorylation.To test, we rst compared the levels of phosphorylated ERK across the intestines in B mice, BC mice, and FBC mice.As anticipated, BRAF V600E increased p-ERK levels throughout the intestine (Fig. 5a).FAK is positively involved in ERK1/2 activation 24 .Consistent with this, in FBC mice, FAK deletion suppressed mutant BRAF-induced elevation of p-ERK (Fig. 5a).The decoupling of ERK pathway output (no change) and the level of p-ERK (reduced) upon Fak loss is in line with a prior report suggesting that the level of ERK phosphorylation does not truthfully re ect ERK pathway activation 20 .
We next examined how Fak loss altered BRAF V600E -induced phosphorylation of ERK.A prior study found that FAK promotes EGFR signaling 43 , raising the possibility that FAK regulates ERK phosphorylation through EGFR.We then evaluated Egfr activation (represented by phosphorylated EGFR at tyrosine 1068) in the mice.The results showed that the level of phosphorylated Egfr Y1068 was increased in BC mice throughout the intestine (Fig. 5b).In FBC mice, Fak deletion moderately reduced BRAF V600E -induced Egfr activation (Fig. 5b) and suppressed Egfr downstream signal transduction as evidenced by the decreased levels of phosphorylated c-Raf S338 and MEK1/2 S217/221 in FBC mice (Supplementary Fig. 3a).To validate that EGFR indeed regulates BRAF V600E -induced ERK phosphorylation, we treated BC mice with the EGFR inhibitor erlotinib.Erlotinib treatment, without signi cantly reducing ERK pathway output (Supplementary Fig. 3b), indeed suppressed phosphorylation of C-RAF, MEK, and ERK (Fig. 5c).Of note, Fak deletion had no impact on the level of p-EGFR and p-ERK in control mice (Supplementary Fig. 3c).Inhibition of Fak kinase activity by FAK inhibitor PF-562271 did not affect the phosphorylation of Egfr and ERK (Fig. 5d), implying that the kinase activity of Fak is not involved in the FAK/EGFR/ERK regulation in BRAF V600Einduced serrated tumorigenesis.
FAK complexes with activated EGFR to promote EGFR signaling 43 .We assessed whether FAK interacts with EGFR in BRAF V600E -mutant cells.The results of co-immunoprecipitation using lysates from cecal mucosa con rmed the Fak-Egfr interaction and revealed that the Fak-Egfr interaction was increased in BC mice and inhibition of Egfr appeared not to affect the Fak-Egfr binding (Supplementary Fig. 3d).ERK phosphorylation is refractory to EGFR inhibition in human BRAF V600E -mutant CRC cell lines 22,23 , however, the FAK-EGFR interaction was still detected in HT29 CRC cells and the interaction was not affected by either EGFR inhibition or FAK inhibition (Supplementary Fig. 3e).These results indicated that FAK/EGFR interaction alone is not su cient for FAK getting involved in the regulation of MAPK signaling.
The contradictory results seen in BC mice and human BRAF V600E -mutant CRC cell lines could result from the differences between in vitro culture systems and in vivo.To test, we examined whether inhibition of Egfr leads to ERK inhibition in freshly isolated cecal crypts from BC mice and BC cecal organoids.The results showed that inhibition of Egfr did not reduce ERK phosphorylation, con rming that the contradictory ndings resulted from in vitro and in vivo.We speculate that the lack of certain stromal factors in vitro is responsible for the EGFR's inability to transmit its signal to activate ERK.
Finally, we examined whether and how a reduction in ERK phosphorylation increases Lgr4 expression/stemness.Our results showed that treatment with MEK inhibitor increased the mRNA expression of LGR4 in human BRAF V600E -mutant CRC HT29 cells (Fig 5f) and BC mice (Fig. 5g), uncovering a negative association between the level of ERK phosphorylation and mRNA expression of Lgr4.Of note, inhibition of ERK activation in BC mice was con rmed by the abrogation of ERK phosphorylation (Fig 5g) and suppression of ERK pathway transcriptional output (Supplementary Fig. 4).This negative association was further supported by our observation that the mRNA levels of Lgr4 were higher, albeit not statistically signi cant, in FBC mice than in BC mice (Fig 5h).Regulation of Lgr4 protein stability represents an important mechanism of modulating Lgr4 function 44 .Our cycloheximide chase analysis results showed that inhibition of ERK phosphorylation by MEK inhibitor treatment dramatically enhanced Lgr4 protein stability in BRAF V600E -mutant CRC cell line HT29 cells (Fig 5i).This nding revealed the inverse correlation between the level of ERK phosphorylation and the protein stability of Lgr4.These results suggest that Fak loss lowers BRAF V600E -induced ERK phosphorylation to increase Lgr4 mRNA expression and protein stability, thereby enhancing intestinal stemness and cecal tumor formation.
Inhibition of ERK phosphorylation downregulates the level of E3 ubiquitin ligase NEDD4.We next investigated how the reduction of ERK phosphorylation increases Lgr4 stability.The HECT-domain E3 ligases NEDD4 (Neuronal precursor cell developmentally downregulated protein 4) and its homolog NEDD4L can ubiquitinate Lgr4, leading to its degradation 45 .Although the RNA-seq data showed no difference in mRNA expression levels of Nedd4 and Nedd4l in C57, BC, and FBC mice, the protein level of Nedd4, but not Nedd4l, was increased in BC mice then decreased in FBC mice (Fig. 6a).To con rm that loss of ERK phosphorylation mediates the Nedd4 reduction, we treated the BC mice with MEK inhibitor and measured the protein levels of Nedd4 and Nedd4l.As shown in Fig. 6b, MEK inhibitor treatment abrogated ERK phosphorylation and reduced the expression of Nedd4, accompanied by increased Lgr4 level.These data suggested that reduced ERK phosphorylation reduces E3 ligase Nedd4 to increase Lgr4 stability.The decreased ubiquitination of LGR4 was con rmed in HT-29 cells.While treatment with MEK inhibitor inhibited the expression of NEDD4 (Fig. 6c), it greatly reduced the ubiquitination of LGR4 (Fig 6d).Together, these data implied that reduction in ERK phosphorylation reduces the expression of E3 ubiquitin ligase Nedd4 in FBC mice to increase the Lgr4 level.
FAK's in uence on oncogenic MAPK-driven intestinal tumorigenesis depends on FAK's impact on ERK phosphorylation.Fak loss reduced ERK phosphorylation in FBC mice (Fig. 5a) but not in control mice with wild-type BRAF (Supplementary Fig. 3c).To determine whether FAK is involved in other oncogenic MAPKdriven tumors, we generated Vill-Cre;Kras LSL-G12D/+ (KC) mice and Vill-Cre;Kras LSL-G12D/+ ;Fak / (FKC) mice.In KC mice, the endogenous expression of oncogenic Kras induces serrated hyperplasia; however, high ERK activation-induced senescence prevents hyperplasia progression into dysplasia 46 .As shown in Fig. 7a, no tumor was found in KC mice (n=6, 9-months-old) and FKC mice (3-month-old, n=3; 6-month-old, n=3; 9-month-old, n=4).Immunoblotting results con rmed that Fak loss failed to in uence the phosphorylation of Egfr or ERK (Fig. 7b).The co-immunoprecipitation results showed that Fak complexed with Egfr in KC mice similarly as in BC mice (Fig. 7c), implying that the noninvolvement of Fak was not due to the lack of Fak/Egfr interaction.A recent preprint (https://doi.org/10.1101/2020.07.02.185173) suggests that "EGFR network oncogenesis cooperates with weak oncogenes in the MAPK pathway", which inspired us to propose the notion that EGFR participates in the regulation of ERK phosphorylation only when the p-ERK level is relatively low.In KC mice, KRAS G12D induces extremely high levels of ERK phosphorylation, high enough to cause intestinal senescence 46 .Given the level of increased p-ERK in KC mice, one would expect that ERK phosphorylation is EGFR-independent.The EGFR independence was con rmed by our results showing that pharmacologic abrogation of EGFR activation had no impact on KRAS G12D -induced ERK phosphorylation in KC mice (Fig 7d).Clinical ndings further supported our notion.Anti-EGFR therapy is excluded for patients with KRAS-mutant CRC, supporting that EGFR has minimum impact on downstream MAPK signaling upon KRAS mutation.However, when ERK activation is inhibited by KRAS G12C inhibitors, EGFR signaling acts as the dominant mechanism of colorectal cancer resistance to KRAS G12C inhibitors 47 .
To address whether FAK downregulation is speci c to human BRAF-mutant CRCs, we compared FAK expression levels in CRCs with different driver mutations using the TCGA database.TCGA analysis revealed that FAK mRNA levels were signi cantly lower in BRAF-mutated CRCs than in APC-mutated CRCs or KRAS-mutant CRCs (Fig. 7e).This result is consistent with the result seen in mice, again, it suggests that FAK is not involved in the regulation of KRAS-mutant CRCs.
In mice, mutant BRAF-induced ERK activation is cancer stage-dependent with signi cantly higher levels of phosphorylated ERK in high-grade dysplasia and carcinoma 3 , suggesting that different tumor stages may require different levels of p-ERK.If FAK is a key regulator of ERK phosphorylation in mutant BRAFinduced serrated tumorigenesis in patients, one would expect the level of FAK may increase as the tumors progress.Consistent with this notion, we observed that FAK levels were higher in BRAF-mutant CRCs than in BRAF-mutant polyps (Fig 1a), TCGA analysis (Fig. 7e) further con rmed that FAK expression was restored to a level similar to normal intestines, albeit still signi cantly lower than in APC mutant or KRAS mutant CRCs (Fig. 7e).
In patients, BRAF mutations are divided into two groups: Activator and ampli er mutation 48 .In CRC, the majority (80%-90%) of activating mutations in BRAF are V600E 18 .Among these mutants, based on their kinase activities, BRAF V600E belongs to the high-activity mutants, and the rest of the mutants except G595R (with impaired BRAF kinase activity in vitro but still induce constitutive ERK activation in vivo) are intermediate activity mutants 49 .If mutant BRAF-induced ERK phosphorylation needs to reach a "justright" level via FAK downregulation in patients, one would expect that the degree of FAK downregulation is BRAF mutant activity-dependent, and there could be a correlation between the activity of BRAF mutants and the degree of FAK reduction.Consistent with this speculation, TCGA data analysis con rmed that CRCs with BRAF V600E mutation had lower FAK expression than CRCs with non-V600E mutations and BRAF wild-type CRCs (Fig. 7f).Although the differences between V600E and non-V600E groups were not statistically signi cant due to limited sample numbers, they might be biologically relevant.

Discussion
The current study nds that in BRAF V600E -mutant intestinal epithelium, elevating the p-ERK level to a minimum threshold is su cient to maximize the pathway transcriptional output, i.e., only lowering the p-ERK level below the threshold will signi cantly abrogate the ERK pathway transcriptional output.Due to the negative association between ERK phosphorylation and intestinal stemness, any increase in ERK phosphorylation will decrease intestinal stemness (Fig 6g).In BRAF V600E -mutant intestinal epithelium, ERK phosphorylation is EGFR/RAS/c-RAF-dependent.The involvement of EGFR provides an opportunity for non-MAPK pathway factors such as FAK to participate in the regulation of ERK phosphorylation to in uence the biological outcomes of BRAF mutation.This study has established the rst "just-right" MAPK signaling model of BRAF V600E -induced tumor formation (Fig. 7g).Our results show that by lowering BRAF V600E -induced ERK phosphorylation, Fak loss, without jeopardizing the ERK pathway transcriptional output, enhances mRNA expression and protein stability of Lgr4, thereby increasing intestinal stemness and promoting cecal tumor formation in mice.
High-level activation of oncogenes (e.g., KRAS, BRAF, and c-MYC) triggers intrinsic tumor suppression 46,[50][51][52][53] .Genetic abrogation of tumor suppressors such as p53 or p16 revokes the tumorsuppressive barrier, thereby facilitating oncogene-induced tumorigenesis 4,46,51,52 .Cooperation with other oncogenic stimulation, such as co-expression of c-MYC and KRAS, ultraviolet radiation on melanocytes expressing BRAF V600E , can also break the suppressive barrier 54,55 .In cellular models 56,57 , overexpression of MKP/DUSPs evades high ERK activation-induced tumor suppression.Whether and how the suppressive barrier can be avoided or reduced in vivo has never been experimentally tested.The current study is the rst demonstration that mutant BRAF-induced activation of ERK signaling is tuneable in vivo, and by tuning ERK activation to alter the suppressive barrier, FAK regulates BRAF transforming activity.
In BRAF-mutated melanoma, a complete shutdown of the MAPK pathway is necessary for signi cant tumor response 58 .In patients with BRAF V600E -mutated CRCs, a combination of encorafenib, cetuximab, and binimetinib (MEK inhibitor) treatment increased the response rate to 26% 59 , highlighting the importance of complete ERK pathway inhibition.However, the inverse correlation between the level of phosphorylated ERK and the level of stemness/Lgr4 expression seen in mutant BRAF expressing intestinal epithelial cells let us speculate that inhibition of ERK phosphorylation may cause stemness increases in BRAF-mutated CRC cells.The molecular mechanisms underlying ERK phosphorylation inhibition-mediated stemness increase remain to be determined.Given the importance of cancer cell stemness in treatment resistance 60 , we propose that the optimal treatment outcome can only be achieved when the inhibition of ERK phosphorylation-mediated stemness increase is simultaneously suppressed.
In sum, the current study reveals the existence of a balance-between the level of phosphorylated ERK, the level of ERK pathway output, and the level of intestinal stemness.Our results show that the "just-right" balance optimal for BRAF V600E -induced cecal tumor formation can be achieved through FAK alteration.Achieving optimal treatment response in BRAF-mutated CRC patients, though, may require abrogation of the p-ERK-stemness regulatory link.That said, the current study could have profound implications for the development of new anticancer agents and new treatment approaches for patients with BRAF-mutated CRC.

Methods
Mice and Treatment.All animal procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee at the University of Pittsburgh.Mice were fed a standard diet (diet ID 5P75; Purina LabDiet, St. Louis, MO).Fak / mice were received from the Mutant Mouse Resource & Research Centers (MMRRC, cat.no.009967-UCD).Villin-Cre (cat.no.021504), Braf LSL-V600E/+ (cat.no.017837), Kras LSL-G12D/+ (cat.no.008179) and Rosa26-tdTomato (cat.no.007914) mice were obtained from the Jackson Laboratory.Genotyping was performed according to the protocols provided by MMRRC and the Jackson Laboratory.Villin-Cre and Braf LSL-V600E/+ mice were crossed to get the BC mice.The littermates harboring Braf LSL-V600E allele were used as controls whenever available.To get the FBC mice, Fak / mice were rst crossed with Villin-Cre mice and Braf LSL-V600E/+ mice, respectively.The offspring Villin-Cre;Fak /+ and Braf LSL-V600E/+ ;Fak /+ mice were further crossed with Fak / mice to get the Villin-Cre;Fak / (FC) and Braf LSL-V600E/+ ;Fak / (FB) mice.The FBC mice were nally obtained by crossing FC and FB mice.The same strategy was used to generate the FKC mice.BC, FBC, KC and FKC mice were euthanized at the indicated age to evaluate the tumor formation.Villin-Cre mice and Rosa26 LSL- tdTomato/LSL-tdTomato mice were crossed to get the Villin-Cre; Rosa26 LSL-tdTomato/+ mice.
For Bromodeoxyuridine (BrdU) labeling, six-week-old mice were given BrdU (MilliporeSigma) at a dose of 100 mg/kg by intraperitoneal injection two hours prior to harvesting.For inhibitor treatment, six-week-old mice were given vehicle (a mixture of 50% DMSO and 50% PEG 400), PF-562271 (60 mg/kg in vehicle) or Erlotinib (100 mg/kg in the vehicle) by a single oral gavage four hours (for immunoblotting) or six hours (for qRT-PCR analysis of ERK output genes) before harvesting.MEK inhibitor PD0325901 was given to mice by oral gavage at a dose of 25 mg/kg in the vehicle.All experiments were performed in both male and female mice.
Cell culture and treatment.HT-29 cells were obtained from the American Type Culture Collection (ATCC) and cultured in DMEM supplemented with 5% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin, in a 37°C humidi ed incubator containing 5% CO2.To study the interaction between FAK and EGFR in HT-29 cells, the cells were treated with DMSO, PF-562271 (5 µM) or erlotinib (10 µM) for one hour before harvested for immunoprecipitation.To study the ubiquitination of LGR4, HT-29 cells were treated with DMSO or 10 µM MEK inhibitor PD0325901 for 24 hours.Then 10 µM MG132 was added to the culture medium and incubated for additional 4 hours before harvesting the cells for immunoprecipitation.
Protein stability assay.HT-29 cells were seeded twenty-four hours before the experiments.The cells were treated with 100 µg/ml cycloheximide (Selleck Chemicals), 10 µM MEK inhibitor PD0325901, or their combination as indicated.Then the cells were harvested, and the whole cell lysates were used for immunoblotting.
Organoid culture and treatment.Mouse organoids were isolated according to the published protocol with some modi cations 61 .Brie y, the cecum of the BC mouse was rinsed with cold PBS, cut into small pieces, and washed eight times in cold PBS by gently pipetting.The fragments were incubated in 10 mM EDTA diluted in PBS for 8 minutes in a 37 °C tube rocker.Then the EDTA solution was removed and the tissue was pipetted 10 times in cold PBS.The supernatant was collected and centrifuged at 300 × g for 3 minutes at 4 °C.The cell pellet was washed with DMEM/F-12 medium and centrifuged at 400 × g for 3 minutes at 4 °C.The pellet was resuspended in Cultrex Reduced Growth Factor Basement Membrane Extract, Type R1 (R&D Systems), and seeded into a 24-well plate.Organoids were cultured using Mouse IntestiCult™ Organoid Growth Medium (STEMCELL Technologies) in a 37°C humidi ed incubator containing 5% CO 2 .The medium was changed every other day.For inhibitor experiments, the freshly isolated crypts (one hour after seeding) and organoids ( ve days after seeding) were treated with 10 µM EGFR inhibitor erlotinib and 10 µM MEK inhibitor PD0325901, respectively, for two hours.To isolate protein for immunoblotting after treatment, the crypt cultures were scraped and suspended in 500 µl of TrypLE Express containing 10 µM EGFR inhibitor or 10 µM MEK inhibitor and incubated at a 37°C water bath for 5 minutes with occasional agitation.After the addition of 500 µl of DMEM/F-12 medium, the crypt cultures were centrifuged at 400 × g for 3 minutes at 4 °C.The cell pellets were resuspended in cold PBS and centrifuged again.The nal pellets were lysed in RIPA buffer (Alfa Aesar) supplemented with protease inhibitor and phosphatase inhibitor (Thermo Fisher Scienti c).Crypt cultures treated with DMSO were used as controls.The lysates were quanti ed and resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and blotted with the indicated antibodies.
Immunoblotting and immunoprecipitation.After the mice were euthanized, the entire intestines were immediately removed and rinsed twice with ice-cold PBS.The mucosal layers of the small intestine (about 1 cm length), colon (about 1 cm length), and cecum (entire cecum, without appendix) were harvested by scraping with a blade and all procedures were performed on ice.The freshly collected tissue was lysed in RIPA buffer supplemented with protease inhibitor and phosphatase inhibitor.The lysates were quanti ed and resolved by SDS-PAGE and blotted with the indicated antibodies.SuperSignal Western Blot Enhancer (Thermo Fisher Scienti c) was used to enhance the blotting signal when needed.To detect the interaction between FAK and EGFR, the tissue lysates were pre-cleared with Protein Gsepharose beads at 4°C for 30 min.The cleared lysates were incubated with anti-EGFR antibody conjugated to agarose (Santa Cruz Biotechnology) or anti-HA a nity gel (MilloporeSigma) at 4°C for 4 hr.
The immunoprecipitates were washed three times with lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP40, and 10% Glycerol, and subjected to SDS-PAGE followed by immunoblotting.The same protocol was used for immunoprecipitation experiments with HT-29 cell lysates.The cell lysates precipitated with anti-HA or anti-Flag beads were used as controls.The antibodies used for immunoblotting are shown in Supplementary Table 2.All experiments were independently repeated at least three times.
Immunohistochemistry, in situ hybridization, BrdU staining, TUNEL staining, and histopathology.The deidenti ed human colon tissue samples from BRAF V600E -mutated CRC patients were provided by the University of Pittsburgh School of Medicine, Department of Pathology tissue core.For mouse tissue sections, the mouse intestine was dissected out, rinsed twice with ice-cold PBS, xed overnight in 10% neutral buffered formalin at 4°C, embedded in para n, and nally cut into 5-μm sections.The sections were depara nized in xylenes and rehydrated in graded alcohol solutions, followed by washes in distilled water.Antigen retrieval was performed for 15 minutes in boiling pH 8 EDTA buffer (Abcam).The sections were allowed to cool to room temperature and then washed with PBS.The endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes.After washing with PBS, the sections were blocked with 20% goat serum diluted in PBS for 45 minutes.Sections were then incubated overnight at 4°C in a humidi ed chamber with primary antibodies diluted in 3% BSA.Primary antibodies used in this study are listed in Supplementary Table 2.The sections were washed with PBS and incubated with secondary antibodies for 1 hour at room temperature.Color visualization was performed with 3.3'-diaminobenzidine until the brown color fully developed.The sections were counterstained with hematoxylin, dehydrated, and coverslipped with permanent mounting media.The slides were scanned using the Aperio digital pathology slide scanner (Leica Biosystems).The images were analyzed using Aperio ImageScope software.
In situ hybridization (ISH) was performed using the RNAscope 2.5 HD Reagent Kit-BROWN (Advanced Cell Diagnostics) according to the manufacturer's instructions.The following probes from Advanced Cell Diagnostics were used: Lgr5 (cat.no.312171) and Lgr4 (cat.no.318321).
BrdU staining was performed on formalin-xed para n-embedded (FFPE) tissue sections using a monoclonal anti-BrdU antibody (MilloporeSigma) as described by the manufacturer.For Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining, the FFPE tissue sections were depara nized, treated with proteinase K and labeled using the In Site Cell Death Death Detection Kit POD (MilloporeSigma) according to the manufacturer's instructions.To quantify the results of BrdU, TUNEL and RFP staining, thirty crypts/villi per mouse were scored for three mice in each group.Myeloperoxidase (MPO) was used as the marker for neutrophils.Ten random-chosen 500 µm-length cecum sections were evaluated for each mouse.MPO + cells within the band of lamina propria, immediately beneath and surrounding the crypts, were counted.Three mice in each group were analyzed.H&E-stained intestinal sections were evaluated for tumor stage by a board-certi ed GI pathologist (Dr.SF Kuan).
Quantitative Reverse-transcription PCR analysis.Total RNA was extracted from the mucosal layer of the mouse intestine or HT-29 cells using the RNeasy Mini Kit (Qiagen).The DNase-treated RNA was reversetranscribed using SuperScript III reverse transcriptase (Invitrogen).The PCR reactions were performed on the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories) using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories).The PCR thermal cycle conditions were as follows: denature at 95 °C for 30 s and 40 cycles for 95 °C, 10 s; 60 °C, 30 s.The speci city of the PCR products was determined by the melting curve analysis.β-actin was selected as an internal reference gene.The sequences of PCR primers are shown in Supplementary Table 3.
Senescence-associated (SA) β-Galactosidase Staining.After the mice were euthanized, the cecum was immediately removed and rinsed with ice-cold PBS.The tissues were frozen in dry ice after the excess liquid was carefully removed using lter paper.Then the tissues were embedded in OCT compound and cut into 10-μm sections.The assays were performed using the Senescence β-Galactosidase Staining Kit (Cell Signaling Technology) according to the manufacturer's instructions.The sections were counterstained with hematoxylin before being dehydrated and coverslipped with mounting media.MSI Analysis.The DNA was extracted from FFPE tissue sections using QIAamp DNA FFPE Tissue Kit (Qiagen).Cecal hyperplasia samples were from 6-week-old FBC mice.Cecal tumor samples were from 9-14.5-month-oldFBC mice.Cecal tissue of 6-week-old B mice was used as control.According to a prior report 62 , ve microsatellite repeat markers, Bat24, Bat26, Bat30, Bat37 and Bat64, were used for MSI analysis.PCR ampli cation was carried out in a multiplex reaction using HSTaq polymerase (Takara Bio, Japan), with primer concentrations 0.5 μM.The thermal cycling conditions were as follows: initial denaturation at 95°C for 5 minutes; followed by 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s; then a nal extension step at 68°C for 30 minutes.PCR fragments were analyzed by capillary electrophoresis, ABI3130XL (Life Technologies), and the GeneMapper ID3.2 program (Life Technologies).
Tumor samples with greater or equal 40% MSI were classi ed as MSI-high (MSI-H), less than 40% as MSIlow (MSI-L), and samples without alterations were classi ed as MSS.
RNA-seq and data analysis.RNA was extracted from the cecal tissues of indicated mice using the RNeasy Mini Kit (Qiagen).After DNase I treatment and performing quality control (QC), 200 ng of highquality total RNA was proceeded to library construction.Oligo(dT) magnetic beads were used to isolate mRNA.The mRNA was fragmented randomly by adding fragmentation buffer, then the cDNA was synthesized using mRNA template and random hexamers primer.Short fragments are puri ed and resolved with EB buffer for end repair and single nucleotide A (adenine) addition.After that, the short fragments were connected to sequencing adapters.The double-stranded cDNA library was completed through size selection and PCR enrichment.Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System were used in the quanti cation and quali cation of the sample library.Finally, the quali ed RNA-seq libraries were sequenced using Illumina NovaSeq6000 in CD Genomics (Shirley, NY) after pooling according to its effective concentration and expected data volume.The FastQC tool was used to perform basic statistics on the quality of the raw reads.Sequencing adapters and low-quality data were removed by Cutadapt (version 1.17).The alignment tool Salmon (version 0.13.1) was employed to quantify transcript expression based on mm10 reference genome.Output les from Salmon were imported into R (V.4.2.0) and analyzed by DESeq2 package (V1.36.0) to identify differentially expressed genes.All genes were ranked by log2(fold change) and used to check the gene set enrichment by using clusterPro ler [4] (V.4.4.1) in R. The following gene sets were used: MAPK signature 20 ; intestinal Wnt signature 35 ; cancer YAP/TAZ target gene signature 36 ; intestinal differentiation signature 37 ; intestinal stem cell signature 38 ; the Hallmark In ammatory Response gene set (Broad Institute) 34 ; upregulated fetal spheroid markers 41 ; upregulated and downregulated genes in human SSA/P 31 (only genes in human SSA/Ps with fold increase>2 or fold decrease<-2 with FDR<0.05 were used).Fak loss inhibits ERK phosphorylation and upregulates Lgr4. a and b Immunoblotting analysis of intestinal mucosa lysates from indicated bowel subsites in indicated 6-week-old mice.c Immunoblotting analysis of cecum lysates from 6-week-old BC mice treated with vehicle or EGFR inhibitor erlotinib for 4 hours.Each lane represented a single mouse.d Immunoblotting analysis of cecum lysates from 6-weekold BC mice treated with vehicle or FAK inhibitor PF-562271 for 4 hours.Each lane represented a single mouse.eImmunoblotting analysis of lysates from freshly isolated cecal crypts and cecal organoids treated with DMSO, MEK inhibitor PD0325901, or erlotinib, respectively as described in Method.fqRT-PCR of Lgr4 using lysates from HT-29 cells treated with the vehicle and MEKi for 4 hours.Data presented as mean ± SD (***P<0.001;Student's t-test, two-tailed).g qRT-PCR of Lgr4 using cecum lysates from BC mice treated with vehicle or MEKi for 6 hours.Data presented as mean ± SD (**P<0.01;Student's t-test, two-tailed).Abrogation of ERK phosphorylation at T202/Y204 in the cecum was con rmed by western blot.h qRT-PCR of Lgr4 in cecum from BC and FBC mice (n=3 per group).Data presented as mean ± SD (P value calculated using two-tailed Student's t-test).i Immunoblotting analysis of the lysates from HT-29 cells treated with cycloheximide (100 μg/ml) and/or MEK inhibitor PD0325901 (10 μM) as indicated.

Figures Figure 1 FAK
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